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Testosterone-propionate has been found to impair response of the cardiac capillary bed to exercise. A study was carried out to quantify the short term, 3-weeks, and long-term, 6-week, effects of combined stimuli of testosterone-propionate and muscular exercise on structural response of the cardiac capillary bed and myocyte hypertrophy. Previously the response of cardiomyocytes to anabolic steroids and exercise at the same time has not been measured.

The Merkel cell-neurite complex initiates the perception of touch and mediates Aβ slowly adapting type I responses. Lichen planus is a chronic inflammatory autoimmune disease with T-cell-mediated inflammation, whereas hyperkeratosis is characterized with or without epithelial dysplasia in the oral mucosa. To determine the effects of lichen planus and hyperkeratosis on the Merkel cell-neurite complex, healthy oral mucosal epithelium and lesional oral mucosal epithelium of lichen planus and hyperkeratosis patients were stained by immunohistochemistry (the avidin-biotin-peroxidase complex and double immunofluorescence methods) using pan cytokeratin, cytokeratin 20 (K20, a Merkel cell marker), and neurofilament 200 (NF200, a myelinated Aβ- and Aδ-nerve fibre marker) antibodies. NF200-immunoreactive (ir) nerve fibres in healthy tissues and in the lesional oral mucosa epithelium of lichen planus and hyperkeratosis were counted and statistically analysed. In the healthy oral mucosa, K20-positive Merkel cells with and without close association to the intraepithelial NF200-ir nerve fibres were detected. In the lesional oral mucosa of lichen planus and hyperkeratosis patients, extremely rare NF200-ir nerve fibres were detected only in the lamina propria. Compared with healthy tissues, lichen planus and hyperkeratosis tissues had significantly decreased numbers of NF200-ir nerve fibres in the oral mucosal epithelium. Lichen planus and hyperkeratosis were associated with the absence of Aβ-nerve endings in the oral mucosal epithelium. Thus, we conclude that mechanosensation mediated by the Merkel cell-neurite complex in the oral mucosal epithelium is impaired in lichen planus and hyperkeratosis.

Electrical potentials up to 800 mV can be observed between different metallic dental restorations. These potentials produce fields in the mouth that may interfere with microbial communities. The present study focuses on the impact of different electric field strengths (EFS) on the growth of Staphylococcus aureus (ATCC 25923) and Escherichia coli (ATCC 25922) in vitro. Cultures of S. aureus and E. coli in fluid and gel medium were exposed to different EFS. Effects were determined by calculation of viable counts and measurement of inhibition zones. In gel medium, anodic inhibition zones for S. aureus were larger than those for E. coli at all field strength levels. In fluid medium, the maximum decrease in the viable count of S. aureus cells was at 10 V⋅m−1. Field-treated S. aureus cells presented ruptured cell walls and disintegrated cytoplasm. Conclusively, S. aureus is more sensitive to increasing electric field strength than E. coli.

Inflammation conveys the development of glomerular injury and is a major cause of progressive kidney disease. NF-κB signaling is among the most important regulators of proinflammatory signaling. Its role in podocytes, the epithelial cells at the kidney filtration barrier, is poorly understood. Here, we inhibited NF-κB signaling in podocytes by specific ablation of the NF-κB essential modulator (NEMO, IKKγ). Podocyte-specific NEMO-deficient mice (NEMOpko) were viable and did not show proteinuria or overt changes in kidney morphology. After induction of glomerulonephritis, both NEMOpkoand control mice developed significant proteinuria. However, NEMOpkomice recovered much faster, showing rapid remission of proteinuria and restoration of podocyte morphology. Interestingly, quantification of infiltrating macrophages, T-lymphocytes, and granulocytes at day 7revealed no significant difference between wild-type and NEMOpko. To further investigate the underlying mechanisms, we created a stable NEMO knockdown mouse podocyte cell line. Again, no overt changes in morphology were observed. Translocation of NF-κB to the nucleus after stimulation with TNFα or IL-1 was sufficiently inhibited. Moreover, secretion of proinflammatory chemokines from podocytes after stimulation with TNFα or IL-1 was significantly reduced in NEMO-deficient podocytes and in glomerular samples obtained at day 7after induction of nephrotoxic nephritis. Collectively, these results show that proinflammatory activity of NF-κB in podocytes aggravates proteinuria in experimental glomerulonephritis in mice. Based on these data, it may be speculated that immunosuppressive drugs may not only target professional immune cells but also podocytes directly to convey their beneficial effects in various types of glomerulonephritis.

For the evaluation of a possible adenovirus-mediated gene transfer into cells of the CNS a replication deficient 3 generation gutless adenovirus vector was used. For cell transduction, vectors with different reporter sequences were applied, resulting in the possibility of measuring the transduction success after infection by the transgene expression. The reporter genes include the green fluorescent protein (GFP), the LacZ (β-galactosidase) as well as the human secreted placenta alkaline phosphatase (SEAP). Applying various infectious particles (10, 50 and 100 MIO) a concentration-dependent transduction rate can be ascertained. The vector was injected into different brain areas including the ventricles. Applying immunohistochemical techniques using antibodies against NeuN (neuronal specific nuclear protein) and the gilal fibrillary acidic protein (GFAP) it can be shown that after vector administration into the striatum a preferential transduction of astrocytes is achieved. Within this brain compartment a diffusion of the vector up to a distance of 1000 ..m from the injection site can be determined. Following injection of vector particles into the corpus callosum, a vector distribution within the entire fibre tract of the white substance including the contralateral hemisphere is observed. With these results it can be shown that the allocation of the vector is dependent on the structure and features of the receiving tissue. Injecting the adenovirus vector into the cerebrospinal fluid results in transgene expression (GFP, LacZ and SEAP) in the ependymal cells surrounding the entire ventricle.After installing a permanent catheter in the lateral ventricle cerebrospinal fluid may be removed in a continuous manner in order to analyse/evaluate the marker enzyme SEAP. Using a luminometric screening procedure, the enzyme SEAP can be demonstrated up to a period of 42 days following vector injection into the cerebrospinal fluid. By these data it can be shown that the use of an adenovirus vector at least under experimental circumstances might be a pioneer therapeutical option for diseases of the central nervous system.

Background:The extracellular signal‐regulated kinases 1 and 2 (ERK1/2) have been implicated in the inflammation‐dependent sensitization of nociceptors. Because the periodontal ligament (PDL) contains numerous nociceptors and mechanoceptors, phosphorylation of ERK1/2 was investigated in nerve fibers of the PDL to elucidate the role of constitutive local activation of ERK1/2 in peripheral sensitization. Methods:Decalcified free‐floating sections of rat molars with PDL were incubated using total (t)‐ERK1/2 and phosphorylated (p)‐ERK1/2 antibodies. For identification of nerve fibers in the PDL, double staining was performed using protein gene product 9.5 (PGP 9.5) with p‐ERK1/2. To test whether p‐ERK1/2 activated in sensory and mechanoreceptive terminals, double incubations were performed using p‐ERK1/2 with calcitonin gene‐related peptide (CGRP) and with calretinin. Labeled nerve fibers were quantified by the point‐counting method. Results:In cervical, midroot, and apical zones of the PDL, t‐ERK1/2– and p‐ERK1/2–labeled nerve fibers were found in close association with blood vessels. The p‐ERK1/2–labeled free nerve fibers were often detected in cervical and apical areas of the PDL. In nerve fibers of the PDL, p‐ERK1/2 was colocalized with PGP 9.5, CGRP, and calretinin. Conclusions:The perivascular distribution of t‐ERK1/2 and p‐ERK1/2 in nerve fibers in the PDL is compatible with a role for the constitutive activation of ERK1/2 in the neural regulation of blood vessels in the PDL. The colocalizations of p‐ERK1/2 with CGRP and calretinin indicate that ERK1/2 is constitutively activated in a subpopulation of sensory and mechanoreceptive nerve terminals in the PDL.

Background:The epithelial rests of Malassez (ERM) are derived from the Hertwig's epithelial root sheath during tooth development. The ERM contain endothelial nitric oxide synthase (eNOS), but the existence of phosphorylation site/s of eNOS in the ERM is unclear. Methods:Rat molars with periodontium were perfusion‐ and post‐fixed, decalcified, and frozen‐sectioned. Free‐floating sections were incubated using antisera against total eNOS, eNOS phosphorylated at serine (Ser)1177, Ser116, and threonine (Thr)495. The signal intensities of t‐eNOS, p‐eNOS at Ser1177and Ser116in the ERM were measured by densitometry and statistically analyzed. Results:In the ERM, localization of total eNOS and the phosphorylation sites of eNOS at Ser1177and Ser116were detected, while a basal localization of eNOS phosphorylated at Thr495in the ERM was undetectable. For p‐eNOS at Ser116regional differences in phosphorylation were detected. Conclusions:The basal production of NO by eNOS in the ERM is modulated by phosphorylation of eNOS at Ser1177and Ser116residues, while the basal activity of the eNOS is not influenced by phosphorylation of eNOS at Thr495residue. This provides evidence that phosphorylation plays a key role for regulation of the catalytic activity of eNOS. J Periodontol 2005;76:1513‐1519.

Endostatin was described as an anti-angiogenic factor. Therefore endostatin looked to be a new way in anti-angiogenic treatment of cancer. Unfortunately, up to now no objective response were seen in clinical trials using endostatin. We compared two different endothelial cell types. Human umbilical vein endothelial cells (HUVEC) and endothelial cells derived from differentiated embryonic stem cells (eESC) were tested in view of endostatin induced proliferation, apoptosis, migration and endostatin binding. Both endothelial cell types had shown an opposite response to endostatin for all observed parameters in dependency of the used concentration. The quantity of HUVEC cells was slightly reduced to 84±8% by treating with 50 ng/ml endostatin whereas the eESC’s showed a significant increase up to 142±12% under same conditions (p=0.01). The observation that endostatin is able to evoke nonuniform response for proliferation, cell mount and migration of endothelial cells, with different endostatin binding characteristic, leads to the assumption that endostatin effect is strongly dependent from endothelial cell type. Furthermore the cell biological response at lower concentration on angiogenic eESC gives evidence for an angiogenic modulatory rather than a predicted anti-angiogenic role of endostatin.

Background:Nitric oxide (NO) mediates different cellular functions by activating soluble guanylate cyclase (sGC) that converts guanosine‐5'‐ triphosphate (GTP) to cyclic guanosine‐3', 5'‐monophosphate (cGMP). Membrane‐bound GCs produce cGMP in response to natriuretic peptides in osteoblasts, but neither the NO‐target enzyme sGC, nor the phosphorylation sites of NOS III, nor their regulation by extracellular signalregulated kinases 1 and 2 (ERK1/2) and Akt/protein kinase B (Akt/PKB) in osteoclasts have been established. Methods:Rat molars with periodontium were perfusion‐ and postfixed, decalcified, and frozen‐sectioned. Free‐floating sections were stained using nicotinamide adenine dinucleotide phosphate‐diaphorase (NADPH‐d) and tartrate‐resistant acid phosphatase (TRAP) histochemical techniques and immunoreacted with antisera against NOsynthase (NOS) I‐III, NOS III phoshorylated at Thr495, NOS III phoshorylated at Serine1177(Ser1177), ERK1/2, phosphorylated ERK1/2, Akt/PKB, phosphorylated Akt/PKB, sGC (α2/β1), and cGMP. Results:NADPH‐d staining and immunostaining of NOS I‐III, NOS III phosphorylated at Ser1177, ERK1/2, Akt/PKB, phosphorylated Akt/PKB, sGC (α2and β1‐subunits), and cGMP were detected in osteoclasts. Immunohistochemical reaction products for NOS III phosphorylated at threonine495(Thr495) and phosphorylated ERK1/2 could not be identified in osteoclasts. Comparison of TRAP activity and immunostaining for sGC β1‐subunit revealed that sGC β1‐subunit is only expressed in a subpopulation of osteoclasts. Conclusions:NO is likely to be generated by NOS I and NOS III in osteoclasts. The inconstant expression of NOS II in some osteoclasts may be explained with inducible expression of NOS II upon physiological cell activation. Localization of the sGC α2‐ and β1‐subunits and cGMP in osteoclasts is compatible with an involvement of NO‐sGC signaling in the homeostasis of osteoclasts. The phosphorylation site of NOS III at Ser1177and phosphorylated Akt/PKB are involved in regulation of NO production by NOS III in osteoclasts under basal conditions. J Periodontol 2004;75:1119‐1125.

Several studies have shown antifibrotic effects of angiotensin converting enzyme (ACE) inhibitors as well as of angiotensin receptor 1 (AT1) antagonists, however, prospective trials with clinical end points comparing these effects do not exist. COL4A3-/- mice develop a non-hypertensive progressive renal fibrosis. We used this animal model to compare the potential of ACE inhibitor vs AT1 antagonist to prevent renal fibrosis irrespective of blood pressure-dependent involvement by the renin system.

The proteolytic fragment of collagen XVIII, endostatin, acts as an inhibitor of angiogenesis. To date, only limited knowledge exists on the effects of endostatin on endothelial cells during embryonic development. Therefore, we analysed the role of endostatin on embryonic vasculo‐ and angiogenesis. Endostatin is accumulated in embryonic tissue of mouse embryos. Similarly, in vessels of embryoid bodies (EBs), endostatin and its binding sites are distributed in vessels and sprouting areas. In EBs, endostatin increases endothelial cells (control, 279.3 ± 76.5; 50 ng/ml, 566.3 ± 90.1; 200 ng/ml, 594.5 ± 166.3 tube‐like structures per EB) and endothelial tubes by proliferation (control, 21.4 ± 7.5; 50 ng/ml, 160.2 ± 9.9; 200 ng/ml, 184.2 ± 16.5 Ki67‐positive nuclei per 50 tube‐like structures); it also enhances migration (control, 380.5 ± 159.8 cells; 50 ng/ml, 718.3 ± 251 cells; 200 ng/ml, 706 ± 89.4 cells) and apoptosis (control, 16.8 ± 6.7; 50 ng/ml, 94.4 ± 23.6; 200 ng/ml, 106 ± 42 PARP‐positive nuclei per 50 tube‐like structures). Furthermore, endostatin modulates the morphology of the endothelial tubes by inducing contraction. Endostatin modulates the embryonic vascular development by enhancing proliferation, migration, and apoptosis as well as by regulating morphogenesis. Developmental Dynamics 230:468–480, 2004. © 2004 Wiley‐Liss, Inc.

Coronary oxygen persufflation (COP) has been shown to prolong heart preservation time up to 14 hr in a mature pig model, with excellent recovery after orthotopic transplantation. The aim of the present study was to assess the structural, metabolic, and functional myocardial and endothelial integrity after COP in mature pig hearts.

Primary vascular plexus originate from angioblasts through a process called vasculogenesis. The precise role of basic fibroblast growth factor (bFGF) and the vascular endothelial growth factor (VEGF) are both suggested as key regulators in vasculogenesis is still unclear. This crucial aspect was investigated by using time lapse observation of in vitrogenerated embryonic stem (ES) cell-derived endothelial structures which were recognizable by using the platelet cell adhesion molecule (PECAM-1) driven endothelial-specific expression of the livereporter gene enhanced green fluorescent protein (EGFP). In serum free conditions VEGF led to improved survival of angioblasts and to the formation of primitive endothelial tubes whereas bFGF alone increased their survival. Our study suggests that the complex process of vasculogenesis can be driven by VEGF alone but not by bFGF.

Recent progress in the cardiotypic differentiation of embryonic and somatic stem cells opens novel prospects for the treatment of cardiovascular disorders. The aim of the present study was to develop a novel surgical approach that allows standardized cellular cardiomyoplasty in mouse with low-perioperative mortality.

Nitric oxide (NO) is an important signalling molecule that plays a relevant role in different cell systems, among them the adult heart. The effects of NO are primarily mediated through modulation of Ca2+homeostasis, myofibrillar contractility, and metabolic regulation in cardiomyocytes. Recent evidence also suggests an important role of NO for cardiomyogenesis by modulating proliferation and differentiation and regulating cardiac function. In the embryonic, but also the healthy and diseased, adult mammalian heart, the inducible (iNOS) and the endothelial (eNOS) nitric oxide synthases (NOS) are detected. However, the expression pattern of NO and its function differ during development. Furthermore, under pathophysiological conditions NOS expression can also change and cause impairment of cardiac performance and cytotoxic effects. The present review focuses on the role and function of NO during cardiomyogenesis, the mechanisms responsible for eNOS availability, and the paracrine effects of NO generated by cardiomyocytes. Microsc. Res. Tech. 55:259–269, 2001. © 2001 Wiley‐Liss, Inc.

The Royal College of Surgeons (RCS) rat is an animal model for retinal degeneration such as the age-related macular degeneration. The RCS rat undergoes a progressive retinal degeneration during the early postnatal period. A potential treatment to prevent this retinal degeneration is the transplantation into the subretinal space of cells that would replace functions of the degenerating retinal pigment epithelium (RPE) cells or may form neurotrophic factors. In this study we have investigated the potential of subretinally transplanted embryonic stem cells to prevent the genetically determined photoreceptor cell degeneration in the RCS rat. Embryonic stem cells from the inner cell mass of the mouse blastocyst were allowed to differentiate to neural precursor cells in vitro and were then transplanted into the subretinal space of 20-day-old RCS rats. Transplanted and sham-operated rats were sacrificed 2 months following cell transplantation. The eyes were enucleated and photoreceptor degeneration was quantified by analyzing and determining the thickness of the outer nuclear layer by light and electron microscopy. In the eyes transplanted with embryonic cells up to 8 rows of photoreceptor cell nuclei were observed, whereas in nontreated control eyes the outer nuclear layer had degenerated completely. Transplantation of embryonic stem cells appears to delay photoreceptor cell degeneration in RCS rats.

ABSTRACTThe extracellular matrix protein fibulin-1 is a distinct component of vessel walls and can be associated with other ligands present in basement membranes, microfibrils, and elastic fibers. Its biological role was investigated by the targeted inactivation of the fibulin-1 gene in mice. This led to massive hemorrhages in several tissues starting at midgestation, ultimately resulting in the death of almost all homozygous embryos upon birth. Histological analysis demonstrated dilation and ruptures in the endothelial lining of various small vessels but not in that of larger vessels. Kidneys displayed a distinct malformation of glomeruli and disorganization of podocytes. A delayed development of lung alveoli suggested impairment in lung inflation. Immunohistology demonstrated the absence of fibulin-1 in its typical localizations but no aberrant patterns for several other extracellular matrix proteins. Electron microscopy revealed intact basement membranes but very irregular cytoplasmic processes of capillary endothelial cells in the organs that were most severely affected. Absence of fibulin-1 caused considerable blood loss but did not compromise blood clotting. The data indicate a strong but restricted abnormality in some endothelial compartments which, together with some kidney and lung defects, may be responsible for early death.

The aims of the present study were to establish if myocardial ischemia/reperfusion is associated with altered eNOS activity and if myocardial eNOS detection depends on its activity. We determined detectable eNOS in (1) myocardium of isolated perfused rat hearts subjected to either global or regional ischemia and (2) in left ventricular biopsies from patients undergoing two different methods of myocardial protection (i.e., intermittent cold blood cardioplegia and continuous coronary perfusion with warm, β-blocker-enriched blood) during coronary artery surgery. NOS detection was performed by NADPH-d staining and three eNOS-antibodies against different eNOS epitopes. In addition, activity dependent alteration of detectable eNOS was proofed by bradykinin treatment for 2 to 10 min. Ischemic and receptor mediated eNOS activation increased NADPH-d reactivity and eNOS immunoreaction as measured by antibodies against either amino acids of a central bovine eNOS domain or the human eNOS N-terminal end. In contrast, the antibody against the human eNOS C-terminal end exhibited no alteration of eNOS immunoreaction. The transient eNOS activation was associated with increased cGMP content. In human myocardium subjected to ischemia during cardiac surgery we found that early reperfusion increases eNOS activity. These data demonstrate a strong association between myocardial ischemia/reperfusion and increased eNOS activity as measured by immunocytochemical staining against specific eNOS epitopes. It appears that eNOS activation and subsequent NO release may act as a regulatory system to counter balance the potentially deleterious effects of myocardial ischemia/reperfusion.

Endostatin is a cleavage product of collagen XVIII that strongly inhibits tumor angiogenesis. To determine if endostatin affects other angiogenic processes, we generated full‐thickness excisional wounds on the back of mice that were systemically treated with recombinant murine endostatin. No macroscopic abnormalities of the wound healing process were observed. Histological analysis revealed normal wound contraction and re‐epithelialization, but a slight reduction in granulation tissue formation and reduced matrix deposition at the wound edge. The blood vessel density in the wounds of endostatin‐treated mice was not affected. However, ultrastructural analysis demonstrated severe abnormalities in blood vessel maturation. The wound vessels in the endostatin‐treated mice were narrowed or closed with an irregular luminal surface, resulting in a severe reduction in the number of functional vessels and extravasation of erythrocytes. Endostatin treatment did not affect the expression level and localization of collagen XVIII mRNA and protein. Furthermore, the angiogenesis regulators vascular endothelial growth factor, angiopoietin‐1, and angiopoietin‐2 were normally expressed in the wounds of endostatin‐treated mice. However, expression of the major wound matrix proteins fibronectin and collagens I and III was significantly reduced. This reduction is likely to explain the reduced density of the wound matrix. Our results demonstrate that endostatin treatment reduces the number of functional blood vessels and the matrix density in the granulation tissue, but does not significantly affect the overall wound healing process.

There are remarkable changes of calcium binding proteins and voltage dependent Ca2+channel subtypes during in vitro differentiation of embryonic stem cell derived neurons. To observe these maturation dependent changes neurones were studied using combined immunohistochemical, patch clamp and videomicroscopic time lapse techniques. Embryonic stem cell derived neuronal maturation proceeds from apolar to bi- and multipolar neurones, expressing all Ca2+channel subtypes. There is, however, a clear shift in channel contribution to whole cell current from apolar neurones with mainly N- and L-type channel contribution in favour of P/Q- and R-type participation in bi- and multipolar cells. Expression of the calcium binding protein parvalbumin could be detected in bipolar, while calretinin and calbindin was preferentially found in multipolar neurones. Our data provides new insights into fundamental neurodevelopmental mechanisms related to Ca2+homeostasis, and clarifies contradictory reports on the development of Ca2+channel expression using primary cultures of neurones already committed to certain brain compartments.

This study examined the occurrence of endothelial nitric oxide (NO)-synthase (NOS-III) in terminal mesenteric vessels and the involvement of NO in microvascular permeability. Possible effects were studied in bradykinin (BK)-induced and basal conditions. NOS expression was investigated by using NOS-III immunohistochemistry and nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase histochemistry on the light- and electron-microscopic levels. Permeability was examined in dissected mesenteries of male rats weighing 250-300 g. Tissue treatment was performed with BK (100 nM), sodium nitroprusside (SNP, 1 and 10 μM), L-nitroarginine (L-NA, 300 μM), BK and L-NA, BK and SNP, L-NA and SNP, as well as with BK, SNP (10 μM), and the guanylylcyclase inhibitor ODQ (10 μM), and BK and ODQ alone. Pharmacologically induced permeability changes were studied with fluorescein isothiocyanate (FITC)-dextran 70 kDa as a tracer for macromolecular transport. Video images were analyzed with computer determination of integrated optical density (IOI). Results were statistically verified by analysis of variance and ttest. Microvascular permeability was increased by 168% after BK treatment and was enhanced by NO-synthesis inhibition with L-NA by 607%. However, the NO donor SNP led to a reduced tracer extravasation to 105 and 58%, respectively, an effect blocked by ODQ. Under basal conditions without prior BK induction, L-NA also causes an increase of IOI by 25%, whereas coapplication with SNP resulted in only a 10% increase of permeability. These results point out that NO has a modulatory role for microvascular permeability by supporting the barrier function of the endothelial lining in stimulated and nonstimulated conditions.

Fibrosis, contraction and opacification of the posterior lens capsule after extracapsular cataract extraction, is a frequent complication following cataract surgery. In these cases, cellular proliferation occurs along an intact posterior capsule. We report a case of fibrosis adjacent to the anterior lens capsule, where cellular proliferations and collagen production completely sealed the anterior capsulotomy three months after a routine extracapsular cataract extraction with implantation of a posterior chamber lens. The fibrosis led to contraction of the remainder of the anterior capsule, significantly reducing vision. Examination of the excised material by light and electron microscopy, and immunohistochemistry, revealed cells with features compatible with fibrocytes that did not stain positive for cytokeratin. The cells were situated in a dense collagen matrix. An anterior capsulotomy that is too small prevents sufficient removal of lens epithelium, and may be a risk factor for this complication.

Acute myocardial ischemia was produced in rabbit Langendorff hearts by ligation of the left anterior descending coronary artery for 2 h. This was accompanied by a significant increase in creatine kinase-specific activity of the ischemic myocardium as compared to sham-operated nonischemic controls (p ± 0.05) and a significant decrease (p ± 0.01) in ATP levels from 2.25–0.29 mol/g wet wt. in the nonischemic area to 0.95 ± 0.21 mol/g wet wt in the ischemic area, indicating a considerable degree of tissue damage. There was a decrease in the norepinephrine ratio between infarcted and noninfarcted myocardium from 1.08 ± 0.08 in sham-operated controls to 0.66 ± 0.06 in ischemic hearts (p ± 0.01). Histochemistry revealed a nearly complete loss of fluorescence in perivascular adrenergic nerves in the ischemic area. Infusion of prostacyclin (PGI2) (1.1 nmol min), starting 10 min prior to ischemia, abolished the increase in creatine kinase activity (p ± 0.05) and the decrease in ATP levels of the ischemic myocardium (p ± 0.05). Furthermore, PGI2prevented the ischemia-induced alterations in catecholamine content and the decrease in adrenergic fiber fluorescence. PGI2did not significantly influence myocardial dynamics and oxygen consumption. To determine the effect of PGI2on nerve stimulation-induced catecholamine release, a separate group of Langendorff rabbit hearts with intact right sympathetic nerves were stimulated twice for 1 min at 0 and 13 min. PCI2at 30 nM 3μM had no significant effect on catecholamine overflow when compared to control hearts. It is concluded that PGI2exerts a stabilizing effect on cell membranes that prevents ischemia-induced destruction of adrenergic nerve endings, This cytoprotective effect is restricted to the ischemic area and does not interfere with the physiological nerve-stimulation-induced norepinephrine release.

Perichromatin granules, nematosomes (threadlike bodies), or concentric lamellar bodies were rarely observed in profiles of principal ganglionic neurons in the untreated rat superior cervical ganglion. They were more frequenlty encountered in these neurons following long-term activation of the sympathetic nervous system by intermittent immobilization (6 to 18 12-h periods). The increased number of the described nuclear and cytoplasmic structures following immobilization is documented by morphometric data and discussed in relation to specific chronic neuronal hyperactivity.

This study investigates the ability of iris epithelial cells (IPE) to ingest rod outer segments (ROS) and compares the amount of phagocytosis of porcine RPE and IPE cells by the use of a pH sensitive fluorescent dye (carboxy SNAFL) at the light microscopic level. The dye allowed investigation of ingestion separately from binding of rod outer segments. In a second set of experiments, after exposing ferritin-labeled ROS to the cultured cells, phagosomes were also counted in electron microscopic sections. Additionally immunocytochemical staining was performed with IPE and RPE cells. Both cell types stained positive with polyclonal NaK-ATPase antibodies against the alpha 1 subunit from rat brain and kidney. The epithelial nature of the cultured cells was determined by monoclonal anti-human-cytokeratin antibodies. Moreover, the ultrastructure of the cells revealed high amounts of phagosomes smaller than 1 μm in diameter present in both RPE and IPE cells. The iron label of the phagosomes was determined by EELS spectra taken from individual phagosomes. Electron and light microscopic quantification shows that cultured IPE cells have 64% of the phagocytic capacity of the RPE with respect to phagosomes larger than 1 μm in diameter.

Abstract: Nitric oxide (NO) is an important mediator in the relaxation of cavernosal smooth muscle. The present study examines the existence and location of the constitutive isoform eNOS (endothelial NO synthase) accompanying the already substantiated neurogenic NOS (nNOS) in the human corpus cavernosum of men with and without erectile dysfunction. Activities of NOS enzymes were examined in specimens of 11 potent and nine long-term impotent patients by means of light and electron microscopy using NADPH-diaphorase staining and immunohistochemical eNOS-specific, smooth muscle actin-specific and nNOS-specific markers. Cavernosal smooth muscle shows a distinct expression of eNOS. In contrast to the weaker expression of eNOS and nitrinergic innervation found in larger veins, the small intracavernosal helicine arteries express large quantities of eNOS and possess a dense nitrinergic innervation. Long-term impotent patients display a broad heterogeneity in eNOS expression and nitrinergic innervation while no overall correlation between NOS expression and erectile function was observed. The expression of eNOS indicates eNOS as a main source of NO alongside nNOS. The differentiated localization of eNOS supports at least a role of this isoform in vascular regulation.

To detect whether toxic intraocular effects of mitomycin C (MMC) after extraocular application are long-standing or are reversible over time.

In the Ham-Wistar rat, a paraganglion was found within the vagus nerve at the site of branching of the recurrent laryngeal nerve. Due to its location the name “paraganglion supracardiale vagi” is suggested. Fluorescence microscopy of the paraganglionic cells displays an intense yellow-green fluorescence indicating the presence of biogenic amines. Ultrastructurally, chief cells containing dense-core vesicles form three kinds of synaptic contacts (afferent, efferent and reciprocal) with enlarged, mitochondria-rich nerve endings.

Objectives. Nitric oxide (NO) is synthesized by the enzyme family of NO synthases (NOS) and plays an important role in tumor growth and angiogenesis. NO generation by inducible NOS (iNOS) also influences the cytotoxicity of macrophages and tumor-induced immunosuppression. Before now, the expression of iNOS and constitutive NOS in bladder carcinoma tissue had not been determined. Methods. Bladder carcinoma tissue specimens were procured from 18 patients (mean age 69.7 years) undergoing transurethral resection. In every patient, tumor biopsies were compared with biopsies of benign bladder regions. Histochemical NADPH-diaphorase staining and NOS immunohistochemistry were performed on all tissue specimens. Results. Positive NADPH-diaphorase staining was detected in all sections from bladder carcinoma tissue. NOS immunohistochemistry showed a different pattern. The malignant epithelial cells were highly iNOS positive. Specimens of bladder mucosa outside of the malignant regions showed only a weak positive iNOS immunostaining. The endothelial cells of abundant precapillary vessels in the stroma of bladder tumors showed a highly positive endothelial NOS (eNOS) immunostaining compared with the stroma of nonmalignant bladder tissue. Neuronal NOS immunoreactivity was only found in nitrinergic fibers in the fibromuscular stroma. Conclusions. Bladder carcinoma tissue had a high iNOS content; benign tissue did not. NO generation from iNOS in the malignant epithelium and from eNOS in tumor stroma may play different roles in tumor angiogenesis and tumor-induced immunosuppression.

Subcutaneous injection of β1 integrin-deficient embryonic stem cells in mice causes the formation of teratomas although they occur with a lower frequency and are smaller than wild-type cells. Immunofluorescence analysis of these deficient tumors indicates a disorganized deposition of several basement membrane proteins. This was confirmed by electron microscopy which demonstrated frequent gaps in cell-associated basement membranes or loss of close contacts to the cells. Further aberrant features were multilaminar structures and amorphous deposits, indicating a strong impairment of correct basement membrane assembly. Quantitative radioimmunoassays were used to determine the levels of specific proteins in successive tissue extracts with neutral buffer in the absence and presence of EDTA and with 6 M guanidine. This demonstrated a more than 90% decrease in the content of laminin-1 (α1β1γ1) and a 70% decrease in nidogen in the β1 integrin-deficient teratomas. No significant changes were detected for other matrix proteins (perlecan, fibronectin, fibulins). This selective change impaired the formation of laminin-nidogen complex and enhanced nidogen degradation. Northern blots also demonstrated a distinctly reduced expression of laminin α1, β1, and γ1 chains. Similar reductions were also observed in cultured embryonic stem cells prior to any differentiation. No or only smaller changes were observed for laminin α2 and β2 chain, nidogen, and perlecan mRNA. These data emphasize a distinct role of β1 integrins in the correct assembly of basement membranes which may occur through direct ligand binding and/or regulatory events at the transcriptional level.

The pleiotrophic but overlapping functions of the cytokine family that includes interleukin (IL)-6, IL-11, leukemia inhibitory factor, oncostatin M, ciliary neurotrophic factor, and cardiotrophin 1 are mediated by the cytokine receptor subunit gp130 as the common signal transducer. Although mice lacking individual members of this family display only mild phenotypes, animals lacking gp130 are not viable. To assess the collective role of this cytokine family, we inducibly inactivated gp130 via Cre-loxP–mediated recombination in vivo. Such conditional mutant mice exhibited neurological, cardiac, hematopoietic, immunological, hepatic, and pulmonary defects, demonstrating the widespread importance of gp130-dependent cytokines.

The development of an avascular filtering bleb following trabeculectomy with mitomycin-C is a frequently encountered clinical phenomenon. We studied a small portion of conjunctiva from such a filtering bleb with light and electron microscopy. Examination revealed markedly irregular epithelium. The substantia propria contained only a few cells and no vessels. The basement membrane of the basal layer of the conjunctival epithelial cells was irregular with areas of variable thickness, breaks, and complete absence. These factors may contribute to the long-standing hypotony following trabeculectomy with mitomycin-C seen in a considerable number of patients.

Prolonged postoperative hypotony is a severe complication frequently associated with trabeculectomies performed with a single perioperative application of Mitomycin C. We performed an animal experiment using 8 pigmented rabbits applying different concentrations of Mitomycin C, ranging from 0.05 to 1.0 mg/ml, under the conjunctiva on the intact sclera for 5 minutes. The eyes were examined by light and electron microscopy after 4 weeks. The nerves within the ciliary body in areas adjacent to the region of treatment showed toxic effects related to the concentrations of Mitomycin C applied. In cases where small concentrations were used, only the unmyelinated nerves were damaged with the myelinated remaining intact. In eyes treated with high concentrations, both groups of nerves exhibited severe signs of destruction. This effect is likely to compromise the production of aqueous humor in rabbits, and possibly also in humans.

Cellular interactions with substrata of the microenvironment are one of the major mechanisms for differentiation and morphogenesis. Many of these interactions are mediated via the β1 integrin subfamily of cell surface receptors, which are believed to transduce signals upon cell adhesion. We have used β1 integrin-deficient embryonic stem cells to test their ability to differentiate into cardiac muscle cells. We show here by several approaches that β1 integrin is important for normal cardiogenesis. First, the in vitro differentiation of β1 integrin-deficient embryonic stem cells into cardiac muscle cells is retarded. This is demonstrated by the delayed expression of cardiac muscle-specific genes and action potentials. Second, the specification of cardiac precursor cells into pacemaker-, atrial- and ventricular-like cells is significantly impaired in β1 integrin-deficient cells. The occurrence of atrial- and ventricular-like cells is reduced and transient. Only cells exhibiting pacemaker-like action potentials of high frequency and arrhythmias survive. Third, the sarcomeric architecture is incomplete and disarranged in the absence of β1 integrin. Fourth, β1-deficient embryonic stem cells can contribute to the developing heart in chimaeric mice but many areas with β1-null cells contain cell debris. The number of β1-null cells decreases from prenatal to postnatal stages and is lost completely in 6-month-old hearts. Thus, we conclude that interactions with the extracellular matrix via β1 integrin is necessary for differentiation and the maintenance of a specialized phenotype of cardiac muscle cells.

The cell specific detection of enzyme activation in response to the physiological contractile load within muscle-tendon-bone unit is essential for understanding of the mechanical forces transmission from muscle cells via tendon to the bone. The hypothesis that the physiological mechanical loading regulates activation of Akt1/PKBα at Thr308 and at Ser473 in muscle fibers within muscle-tendon-bone unit was tested using quantitative immunohistochemistry, confocal double fluorescence analysis, and immunoblot analysis. In comparison to the staining intensities in peripheral regions of the muscle fibers, Akt1/PKBα was detected with a higher staining intensity in muscle fibers at the myotendinous junction (MTJ) areas. In muscle fibers at the MTJ areas, Akt1/PKBα is dually phosphorylated at Thr308 and Ser473. The immunohistochemical results were confirmed by immunoblot analysis. We conclude that contractile load generated by masticatory muscles induces local domain-dependent expression of Akt1/PKBα as well as activation by dually phosphorylation at Thr308 and Ser473 in muscle fibers at the MTJ areas within muscle-tendon-bone unit.